Composition for determination of glucose in body fluids



United States Patent 2,990,338 COMPOSITION FOR DETERMINATION OF GLUCQSE IN BODY FLUIDS Jacob John Gibson, 84-13 246th St., Bellerose 26, NY. No Drawing. Filed Nov. 24, 1959, Ser. No. 855,003 14 Claims. (Cl. 195-1035) This invention relates to a composition for the colorimetric determination of the concentration of glucose in a body fluid, especially human blood.

In accordance with this invention there is provided a color changing composition for colorimetric measurement of glucose, comprising an oxidizable chromogenic material, an enzyme which acts on glucose selectively to produce an oxidizing agent capable of oxidizing the chromogenic material and changing its color and an aminocarboxylic sequestrant or chelating agent which inhibits premature reaction of chromogenic material and enzyme.

In recent years enzymatic reactions have been utilized to test for the presence of glucose. Such tests have been particularly useful in diagnosing diabetes and in following the progress of treatment of that disease. Of special utility have been reactions in which an enzyme, such'as glucose oxidase, acts on glucose in the presence of water and atmospheric or dissolved oxygen to convert the glucose to gluconic acid and also to produce hydrogen peroxide. The hydrogen peroxide then reacts with an oxidizable chromogen and changes its color. This reaction may be promoted by the presence of another enzyme which assists the action of the peroxide, e.g., peroxidase. The degree of coloration or color change of a body fluid test sample is a measurement of the quantity of glucose present. By use of an accurate measuring instrument, such as a spectrophotometer or colorimeter, the transmission through the solution of certain wavelengths of light or other suitable electromagnetic radiation can be determined and, by application of Beers law, the concentration of the colored compound and the concentration of the glucose originally present can be ascertained. The enzymatic processes for glucose analysis are accurate, rapid and easy. They are considered to be more precise than the copper or ferricyanide methods earlier employed and require less of the laboratory technicians time to run. They make possible mass diabetic screening techniques utilizing visual observation of color changes or comparison of colors in spot plate tests or small test tubes.

Although, as outlined above, the enzymatic analysis of body fluid for glucose has much to commend it as a quick and accurate procedure, it has been found that combinations of enzyme or enzymes with chromogen, in aqueous solution or mixture, frequently interact undesirably in the time before admixture with a test specimen. For this reason, compositions of this kind have to be used very shortly after manufacture or results of analyses performed with them may be of doubtful validity. The tendency of the enzymes, chromogen and other constituents of these compositions to interact and deteriorate has hitherto prevented successful manufacture of suitable stable reagents of this type for the accurate determination of concentration of blood sugar. However, now by means of this invention solutions of reagent compositions of this kind can be kept for over six months without objectionable decomposition, discoloration, development of turbidity or precipitation of sediment. They may be used immediately after removal from storage, with no preliminary filtration, standardization or other treatment being necessary. If filtered before use such filtration can be effected easily without any blocking of the pores of the paper by diffieultly filterable solids. Also of excellent stability are powder, particulate, tablet and other solid forms of the composition, which may be made and stored for long 2,990,338 Patented June 27, 1961 2 periods of time without the necessity of a hermetic package or the assurance of a siccative atmosphere.

The color developing material of the present composi-' tions is an oxidizable chromogen. Such compounds are usually colorless in the unoxidized form present in the composition and are oxidized to color by hydrogen peroxide under the right conditions. However, this invention is not restricted only to those color developing chromogens and there may also be employed chromogens that change their color, depending on their stage of oxidation or those which become colored when reduced and are decolorized when oxidized. Only relatively minor changes in the analytical procedure are required when these other chromogens are used and because such changes will be obvious and routine to one of skill in the colorimetric art, they will not be described here.

The oxidation of the chromogen may be speeded with the assistance of an enzyme to help promote the activity of the hydrogen peroxide by furthering its rapid decomposition with the release of oxygen. Although such enzyme helps'hasten the color reaction and allows a quicker determination of the quantity of glucose present in the test sample, the color developing reaction may be effected in its absence, albeit more slowly and less efficiently.

Any suitable dye compound may be used as the chromogen but it is preferred that it be one capable of developing a color distinctively different from the natural color of the composition of which it is a part. As examples of these compounds there may be mentioned orthotolidine, orthodianisidine, diaminofluorene and other derivatives of benzidine-like compounds, here designated as diamino diphenyl compounds. Such dyes are preferably in the forms of their hydrochloric acid salts. The best of the diamino diphenyl dyes known at this time is orthotolidine and the dihydrochloride is the preferred form thereof. Orthotolidine develops a blue color upon oxidation and can be quickly and completely oxidized at room temperature by the oxidizing agent liberated by the action of glucose oxidase on glucose, especially when the oxidation reaction is assisted by the presence of an oxidation-promoting enzyme, such as peroxidase. Despite these obviously significant advantages, orthotolidine, the diamino diphenyl dyes and the other oxidizable chromogens, in the presence of oxidation-promoting enzymes, such as glucose oxidase and peroxidase result in compositions which deteriorate rapidly during storage, especially when in aqueous media or in solutions. For reasons that are not entirely clear to the inventor, his compositions do not possess this disadvantage.

Glucose oxidase or glucose aerodehydrogenase, as it is often called, like all enzymes, is specific in its action. Nevertheless, inhibiting substances present in the body fluid tested should be removed before the test and the test sample should then be filtered. In the testing of human blood the proteinaceous fraction is first coagulated and then filtered off to prevent any interference with the colorimetric determination. The specificity of action of glucose oxidase is such that it is ineffective to promote oxidation of sugars other than glucose. It does not cause the production of oxygen or hydrogen peroxide from compounds normally encountered, other than glucose. It is therefore a highly useful specific reagent to test for glucose. Peroxidase or catalase, as indicated by its names, catalyzes the oxidizing action of hydrogen peroxide and quickly causes the oxidation of oxidizable dyes to their colored forms.

A buffer system useful in regulating the acidity of the blood and reagent being interacted is one which produces a pH of 4 to 6, preferably 5, in the colorimetric solution. Such hustlers are well known in the chemical and analytical arts and are usually and most preferably a mixture of a weak acid with a salt which is derived from a weak acid r 3 1 and a strong base. As examples thereof may be mentioned acetic acid-sodium acetate and citric acid-sodium citrate mixtures. Alternatively, under the correct circumstances, the acids or salts alone may be used. Thus, phosphate bulfers, which are mixtures of monosodium dihydrogen phosphate with disodium monohydrogen phosphate, boric acid, phthalate buifers and others may be suitable. Although various buffers are exchangeable to an extent the reaction which produces a color change in the chromogen appears to be substantially promoted by the presence of buifer of the type described above in which the pH is that at which weak acid and salt of that acid with strong base are in equilibrium. It has been verified that such a buffer, e.g., acetic acid-sodium acetate, helps speed the oxidation of the chromogen by the hydrogen peroxide in the presence of peroxidase and does not cause decomposition of the invented compositions during even extended storage. Compositions comprising chromogen and enzyme, preferably also comprising a buffer, are useful in testing body' fluids for glucose but for accuracy and dependability of the determination, they should be made immediately beforeuse. Even under refrigeration these preparations tend to decompose, developing a color or turbidity, which interferes with accurate colorimen'y, and also losing chromogen through interaction or auto-oxidation, thereby causing a loss of sensitivity of the composition. Consequently, these preparations should not be used after more than a few days frozen storage at most. Otherwise it is necessary to filter this solution frequently, resulting in excessive losses of chromogen, as well as an additional undesirable operation. Due to such limitations on their utility, the enzymatic analytical'procedures for measurement of glucose are not as convenient as is desired.

' It has now been found that the incorporation in these analytical compositions of an aminocarboxylic compound having sequestering or chelating properties inhibits premature reaction of chromogen and enzyme. Such activity is apparently not merely due to tying up any heavy metal catalysts present because the decomposition of the analytical preparation and the loss of chromogen takes place even when the solutions are made with deionized water of very great purity, if the analytical compositions do not contain the required sequestrant. Whatever the nature and mechanism of the decomposition reaction may be the presence of the aninocarboxylic compound prevents :it and allows the composition to be used for as much as several months after manufacture, even when stored as an aqueous solution. Of the amino-carboxylic compounds the amino polycarboxylic compounds are preferred and of these the polyamino polycarboxylic compounds are best. These substances will usually have a lower carboxylic acid radical or salt or a plurality of such radicals on an amino nitrogen'or nitrogcns. The lower acid is of five or fewer carbon atoms. Acetic and propionic radicals are most frequently employed and acetic acid salts are most preferred. In the polycarboxylic polyamino compounds the amino nitrogens are joined by lower alkylene stems, as in the case of the carboxylic radicals, of less than six carbon atoms and of straight chain configuration. Usually there will be less thanfour nitrogens in the amino carboxylic compound and generally the centrally positioned nitrogen will have no carboxylic radical. Thus, it is evident that the polyamino polycarboxylic compounds 'will rarely have more than four oarboxylic acid or salt radicals therein.

Exemplary of the polyamino polycarboxylic compounds are ethylene diamine tetraacetic acid, propylene diamine tctrapropionic acid, ethylene diamine triacetic acid, ethylene diamine diacetic acid, ethylene diamine tripropionic acid and amylene diamine tetraacetic acid, to mention only a representative few of these materials. Nitrilotriacetic acid, nitrilodipropionic acid and glutaminic acid are specific compounds which are monoaminocarboxylic and aminopropionic acid, N-methyl glycine and 4 aminoacetic acid are exemplary of the monoamino monocarboxylic acids. Of course, these latter compounds do not possess the sequestering or chelating power of the polyamino polycarboxylic compounds but they may be of use in stabilizing certain chromogen-enzyme mixtures.

The aminocarboxylic compounds mentioned above were described as acids but their various salts are more frequently employed. Most often the sodium and potassium salts are made but other cations which result in a water soluble salt may also be included in the compounds instead of the alkali metals. With the sequestrants containing higher numbers of carboxylic acid or salt groups the degree of acidity of the test composition usually determines the degree of cation substitution. Thus, the tetrasodium salt of ethylene diamine tetraacetic acid may be converted serially to the trisodium salt, the disodium salt and themonosodium salt, before being completely acidified. Therefore any of the above compounds may be used to obtain substantially the same sequestering or chelating efiect and will inhibit decomposition and deterioration of glucose analysis solutions, if the final compositions in which they are incorporated are brought to the same final pH. In experimental work the tetrasodium and disodium salts have been found to be substantially equivalent. A final pH in the 4 to 6 range is usually found most suitable for enzymatic glucose determinations such as those described in this specification.

The amounts and proportionsfof chromogen, enzymes, aminocarboxylic compound, buffer and deionized water employed should be such as will give a quick determination of glucose present in the blood when the test is run at 25 C. and should be such as to enable one to obtain an accurate analysis, usually within about fifteen minutes minutes or thereabout. Within fifteen minutes the chromogen should have developed a color indicative of the quantity of glucose in the blood being analyzed. To obtain such action the chromogen and enzymes should be present in excess, the chromogen present being more than is needed to react with all the hydrogen peroxide rcleased by action of the glucose oxidase on the glucose. Thus, the chromogen is quickly converted to colored form by the hydrogen peroxide quickly released by the rapid oxidation of glucose due to the action of the glucose oxidase. An excess of peroxidase speeds the oxidation of the chromogen by the peroxide.

Within the teaching of the above paragraph a suit-able analytical composition may comprise to 300 parts orthotolidene hydrochloride (as the dihydrochloride), 5 to 25 parts glucose oxidase, 2, to 5 parts peroxidase, 50 to 250 parts of the sodium salt of ethylene diaminetetraacetic acid, 50to 1,000 parts acetic acid and 1,000 to 4,000 parts sodium acetate in a sufiicient quantity of an aqueous medium to satisfactorily dissolve all the constituents. Preferably, this medium comprises distilled or deionized water without any added solvent or other adjuvant. The amounts given are all by weight, as they are in the examples to follow, and the materials described are all pure, the enzymes being of the higher purities mentioned in this specification.

A very great advantage of the invented compositions resides in the inhibition of deterioration of the analytical compositions when stored as aqueous solution. Thus, these compositions may be sold as liquids, ready to use without the necessity of the analyst or technician weighing out all the components. Alternatively the technician can weigh out the formula when it is needed and may save any excess for future use. The only precautionary steps recommended are storage under refrigeration and a preliminary filtration after the solution has been stored for over a month or so. The useful life of these analytical compositions can be further extended by making a dry pack" of the chromogen, enzymes, aminocarboxylic sequestrant and sodium acetate or other buffer salt. To this may be added an acetic acid solution when analyses are to be begun. Of course, variations may be made as oass .5 in the formula of the dry and wet portions of this composition but usually it will be most desirable to pack all normally solid materials together and add to them the liquids when analyses are begun. Another alternative method of increasing shelf life of these analytical kits is by making two solutions, one containing the enzymes, bufier solution and aminocarboxylic compound, while the other contains chromogen in similar concentration of buffer and aminocarboxylic compound. When a reagent is to be prepared small quantities of the liquids may be mixed easily to give a stable clear reagent in small quantity so as to be economical of reagent and leave less reagent to be stored for future use. The aminocarboxylic compound may be in solution with either the enzyme or chromogen since it is stable with either or both of those materials and even seems to contribute to their own individual stabilities, although such activity is not comparable in importance with the unexpectedly greatly beneficial action of the aminocarboxylic compound in inhibiting premature reaction of enzyme and chromogen and in preventing deterioration of the chromogen in such a system.

The following examples are given to illustrate the invention. It will be clear that they are exemplary only and are not to be considered limitative thereof, the only limitations on the scope of the invention being the allowed claims.

Example I Grams Orthotolidine dihydrochloride .230 Glucose oxidase, crude 1 .070 Peroxidase 2 .004

Disodium salt of ethylene diamine tetraacetic acid .100 Sodium acetate 3.8 Acetic acid 1 N 6.0 Deionized water-enough to bring volume of solution to Manufactured by Sigma Chemical 00., St. Louis, Mo.; 1 milligram will cause an oxygen uptake of approximately 15 cubic millimeters per minute at 35 C. and pH 5.1. Crude glucose oxidase is approximately 10% pure.

Approximntely 60 purpurogailin units per milligram.

The l N acetic acid was made by diluting 57 ml. of glacial acetic to one liter. The formula amount of sodium acetate was dissolved in most of the water and in this solution were dissolved the enzymes and the aminocarboxylic compound, followed by addition of the acid. The orthotolidine dihydrochloride was dissolved in a separate portion of the formula amount of water and was filtered, after which it was mixed with the balance of the formula. The total volume was then brought to 100 ml. To assure that the solution would be optically clear and would not contain any undissolved materials or insoluble impurities accompanying the reagent constituents, the final solution was filtered. In many cases the preliminary filtration of the chromogen solution is unnecessary, providing later filtering takes place. After bottling, the solution was stored in the laboratory refrigerator at 40 F. Portions of the mixed reagent were taken at periodic intervals and glucose analyses of blood were made with them. The solutions were found to be of useful stability for six months or more under such conditions and continued to give accurate analytical results all that time. As a precautionary measure the solution was filtered monthly to remove any minor amount of sediment that may be present. There was no significant loss of chromogen or decrease in sensitivity attendant such filtration.

The blood sugar reagent made was at a pH of approximately 5, that of a Walpole buifer, comprising 7 parts 0.2 molar sodium acetate and 3 parts 0.2 molar acetic acid. The reagent was tested against a standard glucose solution containing 5 milligram percent (5 milligrams glucose per 100 milliliters water) of glucose. Equal volumes of such glucose solution and reagent were mixed at 25 C. and after 15 minutes the developed blue 6 color was read with a spectrophotometer (Coleman Junior, Model 6A), using a wavelength of 630 millimicrons. The optical density of the solution read should be 0.25 approximately.

To deter-mine the glucose content of blood a'sampl is taken with a 0.1 ml. capillary tube, diluted to 2 ml. and then made protein-free by the method of Somogyi (treatment with barium hydroxide and zinc sulfate). The coagulum of proteinaceous material, amounting to less than A of the solution volume, is discarded. One milliliter of the clear supernatant fluid is drawn ofi and is mixed with one milliliter of reagent at 25 C. Fifteen minutes later the optical density is read with a spectrophotometer or photoelectric calorimeter at a transmited light wavelength of 620 to 660 millimicrons. At the inception of the test a control glucose solution made at mg. percent concentration is also treated in the same manner and the control is read after the same development period. The blood glucose concentration is found by multiplying the optical density of the blood sample tested by 100 and dividing by the optical density of the control.

In an alternative method 25 lambda (0.025 ml.) of clear blood serum may be mixed with two ml. of reagent and compared against a similar test result with a serum control of known glucose content. For rough approximations, in the absence of a close control glucose concentration can be calculated by reference to one previously run but this method sometimes leads to errors and a simultaneous control should be run whenever possible.

Example II In the formula of Example I the orthotolidine dihydrochloride was replaced by a smaller quantity, 0.01 g. orthodianisidine dihydrochloride. This solution was prepared as in Example I except that the chromogen was first dissolved in acetic acid and the molarity of the final buffer was 0.4 and the pH was 5.0. A blood sugar determination was made using the same technique as in Example I but employing a different filter because of the different color of the chromogen.

This reagent is also stable under refrigeration and allows accurate analyses to be made even after months of storage.

Example III A glucose test reagent like that of Example I was made for even greater storage stability.

Orthotolidine dihydrochloride 2.3 Glucose oxidase (crude) 0.8 Peroxidase 0.04 Disodium ethylene diamine tetraacetic acid 1.0 Sodium acetate 38.0

When about to be used this composition is dissolved in the requisite amount of water, preferably containing the formula amount of water soluble butter acid (preferably acetic acid). The acetic acid solution may be furnished with the powdered solid composition as part of a reagent I not sufiiciently stable.

kit and solid and liquid parts of the'kit may be proweighed to allow the user to mix without weighing. The reagent powder may be packed inany suitable material, preferably aluminum foil or polyethylene film. This is preferably packed in the same box or package used to hold the container for the acid and the acid container may also serve as a mixing and storing vessel for the finished reagent.

Example VI A two-liquid blood sugar detecting and measuring kit with excellent stability on storage is made by utilizing the basic formula of Example'l in liquid form, with the enzymes alone in solution in one part and the balance of the reagent, including the chromogen in another solution. When ready for use the two liquids may be mixed in measured or pie-measured amounts to make the required quantity of reagent.

The accuracy of the blood sugar determinations made with the invented compositions has been verified by repeated observation and blind checks of controlglucose solutions. The Example I formula was tested against glucose solutions made at a multiplicity of concentrations ranging from 25 to 380 mg. percent. These tests were repeated and deviations from accuracy were tabulated. It was found thatrthe enzymatic method used, with the present reagent compositions, is more accurate and consistent than even the most carefully controlled determinations made by the method of Nelson and Somogyi, previously considered the standard. To make certain that the analysis is of the greatest accuracy a few points should be kept in mind The temperature of the reaction mixture should be kept within the range for which the chromogen is best suited. For orthotolidine, in these compositions, the acceptable temperature range is from 20 C. to 26 C. There is a criticality'in this range because above 26 C., e.g., at 28, the color developed is With, orthodianisidine, on the contrary, stable color is obtained up to about 37 C. The time allowed for color development will depend upon the temperature, the higher the temperature within the per missible range, the less time needed. Maximum color development takes 10 minutes to hour and stability lasts about an equal period.

The invention has been described in detail above. Of course many embodiments of the invention and many equivalents of the components of the invented compositions and process for measuring blood sugar content will now be obvious to those of skill in the art.. It is clear that various'suitable aminocarboxylic compounds may be substituted for the ethylene diamine tetraacetic acid compounds specifically shown in the examples. Usually such compounds will contain Only carboxylic or carboxylic salt groups on the amino alkane stem but noninterfering substituents may also be present. Similarly, other suitable aniline dyes and buffer systems are satisfactory, it being only desirable to adjust the analytical procedure to compensate for any differences between these compounds and the specific preferred compounds described in the examples of this specification. If desired, non-interfering adjuvants may be included in this composition for purpose of improvementthereof.

What is claimed is:

1. A color changing composition for colorimetric detection and measurement of glucose comprising an oxidizable chromogenic material, an enzyme which acts on glucose to produce an oxidizing agent capable of oxidizing the chromogenic material and changing its color and a loweralkylene polyamino poly lower carboxylic acid compound sequestrant which inhibits deterioration of chromogen' in the composition before use.

2. A color developing composition for colorimetric detection and measurement of glucose comprising an oxidizablerchromogem'c material, an enzyme which acts on glucose to produce an oxidizing agent capable'of oxidizing the chromogenic material and developing its color and a lower alkylene polyamino poly lower carboxylic;

acid compound sequestrant which inhibits premature reaction of enzyme and chromogen and maintains the color developing composition at its color before development of the color characteristic of the chromogenic material.

3. A color developing composition for colorimetric detection and measurement of glucose comprising an oxidizable chromogenic material, an enzyme which acts on glucose to produce an oxidizing agent capable of oxidizing the chromogenic material and developing its color, another enzyme which promotes oxidation of the chromogenic material by the oxidizing agent and a lower alkylene polyamino poly lower carboxylic acid compound sequestrant which inhibits premature reactions of enzymes and ohromogen and maintains the color developing compo sition at its initial color before development of the color characteristic of the chromogenic material.

4. A color developing composition for colorimetric determination and measurement of glucose in blood com: prising an oxidizable chromogen colored in oxidized form and capable of developing such color in the presence of hydrogen peroxide and oxidation promoting enzyme, an enzyme which promotes oxidation and color development of the chromogen in the presence of hydrogen peroxide, an enzyme which produces hydrogen peroxide by its action on glucose in the presence of oxygen and water and a lower alkylene polyamino poly lower carboxylic acid compound sequestrant which inhibits premature re action of enzymes and chromogcn and maintains the color developing composition substantially colorless during storage before use.

5, A color developing composition for the colorimetric detection and measurement of glucose in blood comprising an oxidizable chromogen colored in oxidized form and capable of developing such color in the presence of hydrogen peroxide and oxidation promoting enzyme, an enzyme which promotes oxidation and color development of the chromogen in the presence of hydrogen peroxide, an enzyme which produces hydrogen peroxide by its action on glucose in the presence of oxygen and water and a lower alkylene polyamino poly lower carboxylic acid compound sequestrant which inhibits premature reaction of enzymes and chromogen and maintains the color develop ing composition substantially colorless during storage before use.

6. A color developing composition for the colorimetric detection and measurement of glucose in blood comprising an oxidizable chromogen which is an aniline derivative capable of developing color in the presence of hydrogen peroxide and an oxidation promoting enzyme such as peroxidase, glucose oxidase to produce hydrogen peroxide by its action on glucose in the presence of oxygen and Water and a lower alkylene polyamino polylower carboxylic acid compound sequestrant which inhibits premature reaction of enzymes and chromogen and maintains the color developing composition substantially colorless during storage before use.

7. A color developing composition for the colorimetric detection and measurement of glucose in blood comprising an oxidizable chrornogen which is a diamino diphenyl compound capable of developing color in the presence of hydrogen peroxide and an oxidation promoting enzyme such as peroxidase, glucose oxidase to produce hydrogen peroxide by its action on glucose in the presence of oxygen and water and a lower alkylene diamiuo polyiower carboxylic acid compound sequestrant which inhibits premature reaction of enzymes and chromogen and maintains the color developing composition substantially colorless during storage before use.

8. A color developing composition for accurate colorimetric measurement of glucose in blood comprising orthotolidine, peroxidase, glucose oxidase and a compound selected from the group consisting of lower alkylene diamine tetraacetic acid and salts thereof, to inhibit premature reaction of enzymes and orthotolidine and maintain the color developing composition substantially colorless in storage before use.

9. A color developing composition for accurate measurement of the glucose content of blood comprising orthotolidine dihydrochloride, peroxidase, glucose oxidase and disodium ethylene diamine tetraacetate, bufiered to maintain the pH of a blood sample from which proteinaoeous coagu-lum has been removed, at 4 to 6.

10. A color developing composition for accurate colorimetric measurement of the glucose content of human blood comprising glucose oxidase in amount sufficient to promote the rapid reaction of oxygen from air with all the glucose of a specimen of blood to be tested after removal of proteinaceous coagulum therefrom, to produce hydrogen peroxide, orthotolidine in amount suflicient to react with all the hydrogen peroxide generated by action of the glucose oxidase on the glucose in the blood sample to develop color, the depth of which is proportional to the amount of glucose in the blood from which coagulated proteinaceous material has been removed, peroxidase in amount suflicient to promote the quick oxidation of the chromogenic material by the hydrogen peroxide, disodium salt of ethylene diamine tetraacetic acid to inhibit premature reaction of chromogen and enzymes and maintain the color developing composition substantially colorless in storage before use and a buffer which is comprised of a weak acid and a salt of a weak acid and a strong base, in proportions and amounts suflicient to buffer the pH of a deproteinized blood sample within the range 4 to 6.

11. A color developing composition according to claim 2 in which the chromogen and aminocorboxylic compound are dissolved in an aqueous medium packed together with a solution of enzyme, the two solutions being separated so that the enzyme and chromogen are not in contact.

12. A color developing composition according to claim 10 comprising about 100 to 300 parts orthotolidine dihydrochloride, 5 to 25 parts glucose oxidase, 2 to 5 parts peroxidase, to 250 parts disodium ethylene diamine tetraacetic acid, 50 to 1,000 parts acetic acid and 1,000 to 4,000 parts sodium acetate in aqueous solution.

13. A color developing composition according to claim 10 comprising about 230 parts orthotolidine dihydrochloride, 7 parts glucose oxidase, 4 parts peroxidase, parts disodium salt of ethylene diamine tetraacetic acid, 400 parts acetic acid and 3800 parts sodium acetate in solution in about 96000 parts water.

14. A method for determining glucose concentration of human blood which comprises taking a small capillary blood sample from a person whose blood sugar content is to be measured, removing the proteinaceous and cellular constituents from the blood, mixing with a portion of the remaining clear blood serum an aqueous solution of oxidizable chromogen which changes color on oxidation, an enzyme which acts on glucose to produce an oxidizing agent capable of oxidizing the chromogen and an aminocarboxylic compound which inhibits deterioration of the chromogen in the composition before use, allowing the mixture to stand for a suitable length of time between 10 minutes and /2 hour at a suitable temperature between 20 C. and 37 C. and then measuring the optical density or transmission of a beam of light of wavelength suitable for determination of the quantity of color present which is characteristic of the oxidized chromogen, from which optical properties the concentration of glucose present in the blood may be determined.

References Cited in the file of this patent UNITED STATES PATENTS 2,848,308 Free Aug. 19, 1958 2,864,844 Davisson Dec. 16, 1958 OTHER REFERENCES Abstract of Papers, American Chemical Society, both meeting Keston, 1956, 31C.

British Medical Journal, Middleton and Grifliths, December 28, 1957, No. 2, pp, 1525-1527. 

1. A COLOR CHANGING COMPOSITION FOR COLORIMETRIC DETECTION AND MEASUREMENT OF GLUCOSE COMPRISING AN OXIDIZABLE CHROMOGENIC MATERIAL, AN ENZYME WHICH ACTS ON GLUCOSE TO PRODUCE AN OXIDIZING AGENT CAPABLE OF OXIDIZING THE CHROMOGENIC MATERIAL AND CHANGING ITS COLOR AND A LOWER ALKYLENE POLYAMINO POLY LOWER CARBOXYLIC ACID COMPOUND SEQUESTRANT WHICH INHIBITS DETERIORATION OF CHROMOGEN IN THE COMPOSITION BEFORE USE. 